KINETICS OF THE HYDROLYSIS OF MONODISPERSED DIHEXANOYLPHOSPHATIDYLCHOLINE CATALYZED BY BOVINE PANCREATIC PHOSPHOLIPASE-A2 - ROLES OF CA2+ BINDING AND IONIZATIONS OF AMINO-ACID-RESIDUES IN THE ACTIVE-SITE

Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25-degrees-C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, K(m) was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activitY, k(cat), obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the, N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated. The present results are very similar to those for a Group I PLA2 from Naja naja atra venom, but are different from those for Group II PLA2s from Agkistrodon halys blomhoffii and Trimeresurus flavoviridis venoms which show a significant Ca2+-dependence of the substrate binding and do not show any participation of the alpha-amino group in the catalysis [Teshima et al. (1989) J. Biochem. 105, 1044-1051 and 106, 518-527].